1. Field of the Invention
The present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns therapeutic proteases and methods for engineering proteases and protein kinases.
2. Description of Related Art
More than 600 proteases have been annotated so far, constituting the largest enzyme family in the human genome (Overall and Blobel, 2007; Marnett and Craik, 2005; Schilling and Overall, 2008). Because of their unique ability to catalyze the hydrolysis of peptide bonds and thus activate or inactivate proteins, proteases have the potential to be used in a number of applications in biotechnology and medicine (Chanalia et al., 2011; Gupta et al., 2002; Craik et al., 2011). For example, on the medical intervention side, recombinant tissue plasminogen activator (rTPA) protease is commonly used to specifically activate plasminogen to plasmin and thereby prevent or reverse clotting in embolic or thrombotic stroke (Collen and Lijnen, 1991). In addition, thrombin, Factor VII, and Factor IX are approved drugs for the therapeutic modulation of thrombosis and haemostasis (Collen and Lijnen, 1991; Craik et al., 2011; Drag and Salvesen, 2010). Looking toward the future, protease therapies can be envisioned that would involve the specific hydrolysis of validated disease targets (Craik et al., 2011). Because they exhibit catalytic turnover, a therapeutic protease would promise a substantially lower required dose compared with an antibody hitting the same target, since an antibody is expected to operate in stoichiometric fashion. Additionally, proteases find numerous applications as reagents in biotechnology, ranging from analytical to preparative biochemistry (Wehr et al., 2006; Gray et al., 2010; Waugh, 2011).
The list of potential practical applications of proteases, however, is severely limited if one is constrained to the catalytic specificities found in naturally occurring proteases. What is needed, therefore, is a general approach to the engineering of protease specificity (Varadarajan et al., 2008; Gray et al., 2010) and activity. Some previous attempts at rationally redesigning proteases for altered specificity based upon swapping binding site regions between known proteases (Hedstrom et al., 1992; Lim et al., 2007; Villa et al., 2003) have been successful. However, this approach cannot be considered general because its utility is confined to known protease specificities among structurally homologous proteins. Some examples of novel protease specificities, including several reports from the inventor's lab, have been achieved using directed evolution approaches (Sellamuthu et al., 2011; Varadarajan et al., 2005; Varadarajan et al., 2008). Despite this notable progress, two substantial challenges continue to frustrate attempts to use directed evolution to engineer proteases in a more general way.
The first challenge is that multiple mutations are often required to alter protease substrate specificity, necessitating the use of large libraries and therefore a high-throughput screen, such as flow cytometry. For example, in the case of the engineered bacterial protease OmpT, changing the P1 specificity (based on the nomenclature of Schechter and Berger (1967)) required screening a library of 2×108 variants and resulted in improved variants with as many as nine mutations in and around the binding pocket (Varadarjan et al., 2009a). The relatively large number of required mutations makes sense because proteases generally take part in extensive interactions with several different amino acids of their substrates and each substrate amino acid binding pocket is comprised of multiple residues (Schechter and Berger, 1967; Hedstrom, 2002). In addition, changing specificity at a given position may require simultaneous mutation of several contacts and sometimes even second shell binding pocket residues.
The second challenge to engineering specificity is that directed evolution of enzyme function commonly leads to enzyme variants displaying relaxed specificity rather than truly altered specificity (Gould and Tawfik, 2005; Aharoni et al., 2005). Engineered proteases with relaxed specificity may not be suitable for applications involving complex systems or mixtures in which the non-specific activity could have deleterious effects, including in vivo medical applications. To solve this problem, Varadarajan and coworkers demonstrated a simultaneous selection and counter selection strategy for protease activity that resulted in protease variants capable of reacting with new substrates, while maintaining a relatively narrow overall substrate specificity (Varadarjan et al., 2005). Their screening method, however, requires display of the protease on the surface of E. coli, thus limiting the method to proteases that can be expressed in E. coli, transported to the surface, and are active in the extracellular environment. Clearly, there is a need for new methods for engineering proteases.